N-nitroso compounds are among the most potent demonstrated carcinogens. Their occurrence in foodstuffs and tobacco has led to speculation that they comprise an important group of carcinogens in man. It is generally accepted that their metabolities are the ultimate carcinogens. This grant proposal addresses itself to the problems of identification and mode of formation of the ultimate carcinogen. Solution of these problems is of importance in recognizing other petential sources of the ultimate carcinogen, and in evaluating factors that may influence the formation of the ultimate carcinogen. As most carcinogens are mutagens, or can be activated to mutagens, the molecular mechanisms responsible for carcinogenicity and mutagenicity are likely to be similar. It is proposed here, to combine an enzymatic metabolizing system of N-nitroso compounds with a mutagenic assay system to investigate the mechanism of mutagenesis by N-nitroso compounds. Several Salmonella tester strains, developed for screening potential carcinogens, will be tested as mutagens. The reaction mix will be quantitatively analyzed for reactant consumption and product formation. Product incorporation into microorganisms and DNA will also be investigated. It is anticipated that hepatic enzymes and microsomes will most frequently be employed as the metabolizing enzyme. Tissue homogenates, microsomes, and enzymes from several other organs will also be investigated. As the metabolizing enzymes themselves may incorporate products and thus complicate product analyses, chemical methods of generating the ultimate mutagen will be explored. To compare in vitro and in vivo metabolism, urine samples from animals treated with N-nitroso compounds will be analyzed for reactants, and products and will be tested for mutagenic activity. Several methods of isolating the ultimate mutagen, or its immediate precursor, will be examined. If one of these proves successful, direct testing of the ultimate mutagen as a carcinogen may be instituted.